Chronic alcohol consumption can cause progressive liver damage, resulting in steatohepatitis and cirrhosis, which can lead to other more fatal complications such as hepatocellular carcinoma (HCC). Progression of alcoholic liver disease (ALD), like many chronic forms of liver disease, is driven by hepatocyte damage that promotes inflammatory responses and the activation of hepatic stellate cells (HSC) into matrix-producing myofibroblasts (MF). Our collaborator has shown that activation of the Hedgehog (Hh) signaling pathway plays a critical role in how damaged livers respond to acute and chronic injuries, including ALD. Activation of matrix- producing MF in damaged livers is considered the critical step in the progression of chronic liver disease to cirrhosis and is a process that is regulated in part by Hh signaling. There is now compelling evidence that the embryonic developmental Hh pathway becomes reactivated during adult liver injury and plays a key role in the wound healing response. Deregulation of the Hh pathway occurs during cirrhosis and in many primary liver cancers. Studies have shown that antagonists of the Smoothened gene (SMO), a component of the Hh pathway, have the potential to treat fatty liver disease and alcohol-induced cirrhosis. In this proposal, RNA silencing of SMO will be explored as a highly specific therapy for ALD. While several companies are attempting to harness the RNA interference (RNAi) process to create potent and specific therapeutics, the critical barrier to progress in the field is the ability to systemically deliver long-lasting RNAi-based therapeutics. Currently, siRNAs are injected intravenously (IV) where they undergo rapid clearance from the body if not formulated with potentially toxic and immunogenic lipid-encapsulating vehicles. In this proposal, we apply our novel carrier technology to a new class of siRNA called sdRNAs, which can efficiently silence genes and are capable of penetrating cells without formulation. Our carrier molecule, when conjugated to peptides and proteins, significantly improved pharmacokinetic (PK) properties by preventing kidney clearance and increasing subcutaneous (SC) bioavailability. Use of this carrier with sdRNAs may allow them to be long-acting and have the potential to be delivered SC rather than IV. If successful, this would be truly enabling for this drug class. In the studies proposed here, modified sdRNAs specific for SMO will be identified, conjugated to the carrier, then examined in vitro to confirm that their cell-penetrating and silencing properties have not been altered following conjugation. The half-life and bioavailability of the sdRNA conjugates will then be tested in a PK study in rats. We expect that these sdRNA conjugates will now be long-acting and able to be systemically delivered, potentially SC. Finally, the sdRNA conjugates will be tested for efficacy in an animal model of ALD. This Phase I proposal aims to demonstrate the feasibility of SMO-specific sdRNA conjugates as an approach to treat alcoholic liver disease. The carrier-based technology can then be applied to other classes of oligonucleotide therapeutics and could be a general approach for in vivo functional genomic and target validation studies.